When (14C) CHC13 was incubated with liver microsomes in the presence of increasing concentrations of cysteine, the covalent binding of the (14C) label to protein and the formation of (14C) CO2 were found to progressively decrease; concomitantly, the formation of (14C) 2-oxothiazolidine-4-carboxylic acid increased. This compound, which is formed from phosgene, COCl2, and cysteine, accounted for approximately 65% of the decreases in binding and liberation of CO2. When (3H) CHCl3 was incubated with liver microsomes, no binding to protein was detected. These results establish that CHCl3 is activated in vitro exclusively by metabolism of the C-H bond, and that metabolic oxidation of this bond to produce COCl2 appears to be a major activation process. Base hydrolysis studies of microsomal protein containing covalently bound (14C) label support this conclusion. BIBLIOGRAPHIC REFERENCE: Sipes, G.L., Krishna, G. and Gillette, J.R.: Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: Role of cytochrome P-450. Life Sci., 20: 1541-1548, 1977.